Abstract Kinetic studies on hydrogen peroxide–induced inactivation of mutant formate dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of hydrogen peroxide, the decrease in enzyme activity follows first–order kinetics. Therefore, the first–order effective inactivation kinetic constants determined for various FDH forms at a constant H2O2 concentration can be used as a quantitative measure of the enzyme stability. It was shown that two cysteine residues located in the active site formate– and coenzyme–binding domains (Cys145 and Cys255, respectively) make similar contributions to the enzyme stability, while the contribution of Cys354 is insignificant. The inactivation kinetics of wild–type PseFDH, mutant PseFDH Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium Staphylococcus aureus, higher plants Arabidopsis thaliana, and soya Glycine max, was studied. It was found that the stress–induced FDHs are at least 20 times more stable than the nonstress–induced PseFDH from Pseudomonas sp. 101 grown on methanol.